For many years, researchers have used spectroscopy and microscopy techniques to identify biological materials within a sample. The techniques typically involve applying a light to a sample and then analyzing the fluorescence light emitted from that sample, where the emitted light may be compared to known characteristic spectra data to identify the biological material in the sample. Traditionally, researchers used these techniques to measure static values, such as the overall concentration of a biological material within a sample.
More recently, as biological study has matured, researchers have developed a need for moving beyond static measurements toward making real-time measurements of intracellular molecular events in living cells. That is, researchers have developed a need for real-time spectroscopy and microscopy techniques. For example, it has become increasingly important to develop non-invasive experimental approaches of monitoring molecular activities of biological complexes in living cells. With real-time measurements researchers could measure such molecular activities, which would help researchers examine sub-micron and nano-metric biological complexes. With nano-metrically sized biological complexes (e.g., clusters of receptors, protein-RNA, protein-DNA associations, RNA-RNA, DNA-DNA and cells), it is desirable to be able to monitor and characterize the associations and functions of isolated biological complexes, and preferably with visually representative data. This desire to monitor associations and functions of isolated biological complexes is not only important in living cells, but is also important for in vitro experiments, such as protein binding assays and RNA or DNA microarrays, for example. Yet, despite the need for monitoring techniques capable of resolving phenomena at molecular or intracellular levels, present spectroscopy and microscopy techniques are insufficient.
Although introduced nearly thirty years ago, researchers have recently started using fluorescence correlation spectroscopy (FCS) to resolve small-scale associations and functions in biological samples. The techniques are capable of fluorescence detection over small detection volumes, approximately the size of E. coli. Fluorescence correlation spectroscopy offers quantitative information by analyzing the spontaneously fluctuating fluorescence intensity obtained from diffusing fluorescent molecular complexes, thus, allowing for real-time measurements.
In implementation, however, these conventional FCS techniques use a wide-field evanescent wave to illuminate a sample. And although some success has been reported, these techniques are limited in the size of the sample being tested. By using wide-field evanescent waves, such as those created by total internal reflection from a transversely-propagating laser beam, overly large sample volumes are illuminated. These volumes result from the disperse intensity profile of wide-field evanescent waves, which exhibit a Gaussian profile. In FCS systems, these large sample volumes present a number of problems for researchers.
One problem is that, because of the overly large FCS detection volumes, the sample volume tested will have a large number of contaminants. Thus, in measuring biological complexes such as membrane protein activities or receptor clusters, these large sample volumes will not only contain the molecules being measured but also large numbers of contaminant molecules, which represent noise in spectroscopy measurements. And in current FCS systems, this noise may be large enough to prevent accurate measurement of biological complexes below a certain dimensional size.
An example conventional FCS technique is that of the ConfoCor 2 system from Carl Zeiss, Inc. of Germany, which measures the concentration and the diffusion of fluorescent molecules using FCS. The FCS detection volume of the ConfoCor 2 system has reported dimensions over a micron in length (1.5 μm) along the Z axis, which means an FCS detection volume that is much larger than the typical size of a protein complex, which is about 100 nm. Such large volume systems are likely to be too noisy for accurate measurements of smaller-sized biological materials.
TIR (total internal reflection) illumination combined with FCS presents a bleaching problem, as well. Bleaching is the destruction of a portion of a sample due to the high intensity of the light source illuminating the sample during spectroscopy. In conventional wide-field TIR the entire field of view is illuminated, which causes bleaching outside the region of detection.